Kaposi's sarcoma (KS)-associated herpesvirus (KSHV/HHV8) is a DNA tumor virus etiologically associated with KS and several other malignancies. The applicant's long-range goal is to use KSHV as a paradigm to understand the molecular mechanism of virus-induced pathogenesis, providing biologic basis for preventive and therapeutic purposes. The overall hypothesis is that KSHV encodes specific genes to evade host anti- viral defenses and complete viral life cycle. An array of KSHV genes has been shown to deregulate cellular functions when cloned and examined individually; however, their precise functions in viral infection and KSHV-related pathogenesis remain unclear. The lack of an efficient infection model has hampered the investigation of KSHV infection, replication and cellular transformation, and genetic analysis so far. We have recently cloned the entire KSHV genome into a bacteria artificial chromosome (BAC) and reconstituted it in 293 cells. Recombinant virions infect can 293 and human primary endothelial cells at ~100% primary infection efficiency, and establish stable latent infection. Infected endothelial cells are transformed into KS- like vascular spindle cells. Recombinant genomes can also be easily shuttled between bacteria and 293 cells. The objective of this application is to develop and characterize cell models for KSHV infection, replication, cellular transformation, and genetic analysis, and to use them to examine functions of viral immediate-early (IE) genes. The central hypothesis is that infection of human primary endothelial and 293 cells with the highly infectious recombinant KSHV closely mimics the natural viral life cycle, and together with the highly efficient bacteria-mammalian (293 cells) shuttle system, could be used as cell models for viral infection and replication, cellular transformation, and functional dissections of viral genes. The rationale is that such cell models are vital for understanding KSHV virology and KSHV-related pathogenesis, and IE genes have critical roles in viral infection and replication. There are three specific aims: 1) To determine the expression patterns of viral and cellular genes, and phenotypic characteristics of recombinant KSHV infection and replication in 293 cells; 2) To determine the virology and cellular biology of recombinant KSHV primary, latent and lytic infection, and cellular transformation of human primary endothelial cells; 3) To define the functions of KSHV IE genes in viral infection, replication, and cellular transformation through characterizing viral mutants using the cell models. The proposed work is innovative, because it will establish the first efficient cell models for KSHV infection, transformation, and genetic manipulation. These studies will be significant, because they will define the virology and cellular biology of KSHV primary, latent and lytic infection, and cellular transformation as well as the functions of IE genes, thus provide insight into the pathogenesis of KSHV-related malignancies. The cell models will also be useful for the development of anti-KSHV vaccines and therapeutic drugs. PERFORMANCESITE(S) (organization,city,state) The University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, Texas 78229-3900 KEYPERSONNEL.See instructions. Use continuationpagesasneeded to providethe requiredinformationin theformat shownbelow. Startwith PrincipalInvestigator.List allotherkey personnelinalphabeticalorder,last namefirst. Name Organization Roleon Project Gao, Shou-Jiang UTHSCSA Principal Investigator Flore, Ornella New York University School of Medicine Scientific Consultant Christopher P. Jenkinson UTHSCSA Scientific Consultant Robert E. Lanford Southwest Foundation for Biomedical Scientific Consultant Research and UTHSCSA Zhang, Yanjin UTHSCSA Postdoctoral Fellow Zhou, Fuchun UTHSCSA Postdoctoral Fellow _ PHS 398(Rev.05/01) Page_2 FormPage2 _ _ Principal Investigator/Program Director (Last, first, middle): GAO, SHOU-JINAG The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ......................................................................................................................................................... 1 Description,